Isolation of cells for in vitro studies or for applications in cellular therapies usually incorporates an initial separation of blood cell components mainly based on the bulk depletion of erythrocytes, which comprise >99% of the cellular mass of blood. Other cell types a so are removed that provide no long-term therapeutic potential (e.g., granulocytes), contribute to pathology (e.g., T-cells in graft versus host disease (GVHD) associated with bone marrow transplant, or erythrocytes in transfusion related reactions), or, in general, interfere with the ability to monitor the cell population of interest. Depletion of T-lymphocytes from bone marrow before implantation is a common technique used to reduce the incidence or degree of GVHD, which is mediated by T-cells. Techniques used to deplete these cell populations differ depending upon the cell population that is to be removed. Complete removal of T-cells may not be desirable as they might contribute to graft vs. tumor effect. A tunable cell separation medium that can be adjusted to remove specific levels of T-cell contamination could be a useful tool for the preparation of allogeneic stem cell transplants.
Techniques for erythrocyte removal are based on hypotonic lysis of erythrocytes, density gradient separation, or enhanced centrifugal sedimentation using hydroxyethyl starch. Hypotonic lysis, while useful in low volume in vitro studies, can be impractical for the large volumes of blood tissues processed for cellular therapies. If utilized in cell therapy procedures, erythrocyte hypotonic lysis usually is done as a final clean-up step to remove the remaining erythrocytes that may contaminate a sample after bulk depletions by other methods.
Density-gradient separation relies on differences in the density of different cell types causing them to segregate at different levels in a fluid medium of variable density. Differences in density between the cell types can be small, and individual cells types can be heterogeneous in size and density. Consequently, particular cell types can become distributed throughout a density-gradient medium rather than precisely segregating at a discrete area in the density medium, resulting in reduced recovery of desired cells and/or contamination with undesired cell types. In procedures that enrich for rare blood cell types such as hematopoietic progenitor cells, density-gradient sedimentation can lead to loss or reduced yields of desired cell subsets. For example, using conventional density-gradient methods to isolate progenitor cells (e.g., CD34+ hematopoietic stem cells) from umbilical cord blood results in a significant loss of the desired stem cells. See e.g., Wagner, J. E., Am J Ped Hematol Oncol 15:169 (1993). As another example, using conventional density-gradient methods to isolate lymphocytes results in selective loss of particular lymphocyte subsets. See e.g., Collins, J Immunol Methods 243:125 (2000). These separation methods have an addition contraindication for use in cellular therapies in that the chemical entities in the separation medium can be toxic if infused with the cells into the recipient. As such, additional steps must be performed to ensure their complete removal prior to infusion. Instrument methodologies such as elutriation also depend upon differential separation of blood components by density and can suffer from similar deficiencies in performance.
An additional method for removing erythrocytes from blood includes mixing with hydroxyethyl starch (i.e., heta starch), which stimulates the formation of erythrocyte aggregates that sediment more rapidly than leukocyte components when sedimented at 50×g in a centrifuge. While this method is generally non-toxic and ‘safe’ for the recipient, its performance in the recovery of important cell types, including, for example, hematopoietic stem cells, is variable depending upon factors such as temperature, age of sample (post-collection) prior to processing, cellularity (concentration of cells per unit volume) of sample, volume of sample, and ratio of anti-coagulant to blood sample. These factors, with respect to umbilical cord blood, for example, can result in less-than-ideal recovery of stem cells and diminution of the engraftment potential of the cord blood cells, increasing the risk for transplant failure.
Increasing the recovery of rare cell types from donor tissue could dramatically improve the outcomes of transplant and immune therapies (e.g., bone marrow transplants, stem cell-based gene therapy, and immune cell therapy), the success of which apparently is related to the actual number of the cells being used for therapy. Additionally, with allogeneic bone marrow or cytokine-elicited stem cell transplants, implants containing the highest possible recovery of stem cells in conjunction with a partial removal of T-cells, may favor the best chance for successful transplant survival.